Evolution of the Nitric Oxide Synthase Family in Vertebrates and Novel

Home , Arowana, Bichir, Cavefish, Chaca (fish), Chaca chaca, Elopomorpha

bioRxiv preprint doi: https://doi.org/10.1101/2021.06.14.448362; this version posted June 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Evolution of the nitric oxide synthase family in vertebrates 2 and novel insights in gill development 3 4 Giovanni Annona1, Iori Sato2, Juan Pascual-Anaya3,†, Ingo Braasch4, Randal Voss5, 5 Jan Stundl6,7,8, Vladimir Soukup6, Shigeru Kuratani2,3, 6 John H. Postlethwait9, Salvatore D’Aniello1,* 7

8 1 Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121, 9 Napoli, Italy 10 2 Laboratory for Evolutionary Morphology, RIKEN Center for Biosystems Dynamics 11 Research (BDR), Kobe, 650-0047, Japan 12 3 Evolutionary Morphology Laboratory, RIKEN Cluster for Pioneering Research (CPR), 2-2- 13 3 Minatojima-minami, Chuo-ku, Kobe, Hyogo, 650-0047, Japan 14 4 Department of Integrative Biology and Program in Ecology, Evolution & Behavior (EEB), 15 Michigan State University, East Lansing, MI 48824, USA 16 5 Department of Neuroscience, Spinal Cord and Brain Injury Research Center, and 17 Ambystoma Genetic Stock Center, University of Kentucky, Lexington, Kentucky, USA 18 6 Department of Zoology, Faculty of Science, Charles University in Prague, Prague, Czech 19 Republic 20 7 Division of Biology and Biological Engineering, California Institute of Technology, 21 Pasadena, CA, USA 22 8 South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, 23 Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceske 24 Budejovice, Vodnany, Czech Republic 25 9 Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA 26 † Present address: Department of Animal Biology, Faculty of Sciences, University of 27 Málaga; and Andalusian Centre for Nanomedicine and Biotechnology (BIONAND), 28 Málaga, Spain 29 30 * Correspondence: [email protected]

33 ORCID

34 GA: 0000-0001-7806-6761

35 JPA: 0000-0003-3429-9453

36 IB: 0000-0003-4766-611X

37 JS: 0000-0002-3740-3378

38 VS: 0000-0002-1914-283X

39 SK: 0000-0001-9717-7221

40 JHP: 0000-0002-5476-2137

41 SDA: 0000-0001-7294-1465

58 Abstract

59 Nitric oxide (NO) is an ancestral key signaling molecule essential for life and has

60 enormous versatility in biological systems, including cardiovascular homeostasis,

61 neurotransmission, and immunity. Although our knowledge of nitric oxide synthases (Nos),

62 the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified

63 repertoire of nos gene orthologs in fish with respect to tetrapods remains a puzzle. The

64 recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in

65 the ray-finned fish lineage, changed this perspective. This prompted us to explore nos

66 gene evolution and expression in depth, surveying vertebrate species representing key

67 evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced

68 several lineage-specific gene duplications and losses. Second, nos3 was found to be lost

69 independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third,

70 the expression of at least one nos paralog in the gills of developing shark, bichir, sturgeon,

71 and gar but not in arctic lamprey, suggest that nos expression in this organ likely arose in

72 the last common ancestor of gnathostomes. These results provide a framework for

73 continuing research on nos genes’ roles, highlighting subfunctionalization and reciprocal

74 loss of function that occurred in different lineages during vertebrate genome duplications.

81 Keywords: Vertebrate evolution; Genome duplication; Gene duplication and loss; NOS;

82 Phylogenomics; Synteny.

83 Introduction

84 Originally classified as a pollutant, nitric oxide (NO) was recognized as “Molecule of the

85 Year" in 1992 [1] when its important role as a cellular signaling molecule was recognized.

86 NO plays a role in a myriad of physiological processes, such as cardiovascular

87 homeostasis [2], neurotransmission [3], immune response [4], and in pathological

88 conditions such as neurodegenerative diseases [5] and cancer [6].

89 Nitric oxide synthase (Nos), the enzyme catalysing the biosynthesis of NO in vivo, is

90 ubiquitous among organisms, including protists and bacteria [7,8]. Three nos gene

91 paralogs have been described in vertebrates: two constitutively expressed genes,

92 including nos1 (also known as neuronal nos, or nNos), which represents the predominant

93 source of NO involved in neurogenesis and neurotransmission [9,10], and nos3

94 (endothelial nos or eNos) implicated in angiogenesis and blood pressure control in

95 vascular endothelial cells [11,12]. In addition, nos2 (inducible nos or iNos), which

96 expression is instead evoked by proinflammatory cytokines, is promptly activated in a

97 range of acute stress responses [13].

98 Although the availability of current genomic data covers all major ray-finned fish lineages,

99 the evolutionary history of their nos gene repertoire remains puzzling. Previous studies

100 reported a variable number of nos genes in teleost fishes: nos1 is always present in a

101 single copy; nos2 either in one or two copies, probably due to the additional teleost-

102 specific whole-genome duplication (TGD) [14–17], or absent as observed in several

103 species. On the other hand, nos3 has been reported as missing in the genomes of ray-

104 finned fish. This apparent gene loss contrasts with literature describing a putative Nos3-

105 like protein localized by antibody stains in gills and vascular endothelium of several teleost

106 species [18,19]. The discovery of a nos3 ortholog in the spotted gar Lepisosteus oculatus,

107 a holostean fish (the sister group of teleosts within the ray-finned lineage) [20], and the

108 variable number of teleost nos2 genes raises new questions about the evolution of this

109 important gene family, including: i. is our current view on the origin and evolution of nos

110 gene family in vertebrates accurate?; and ii. can further investigation of nos expression

111 pattern in fish retaining a nos3 copy reveal novel functional insights? In an attempt to

112 answer these questions, we have studied the Nos family repertoire at unprecented

113 phylogenetic resolution, investigated conserved syntenies in fish genomes, and studied

114 the expression pattern of all three nos genes during development in multiple species

115 representing key nodes in vertebrate evolution.

116

117 Results

118 Revised evolutionary history of Nos2 and Nos3

119 Gaps in our current knowledge of Nos family evolution include the time of origin of the

120 three distinct paralogous nos genes and when some of them were secondarily lost in

121 specific lineages. Using sequences retrieved from public genomic and transcriptomic

122 databases, we reconstructed a Nos phylogeny using 108 protein sequences from 53

123 species (see Supplementary Table 1). Species were chosen to provide a broad

124 representation of aquatic vertebrates: cyclostomes (modern jawless fish), chondrichthyans

125 (cartilaginous fish), and osteichthyes (bony fish) including ray- and lobe-finned fishes.

126 Lobe-finned fishes include coelacanths, lungfishes, and tetrapods; Ray-finned fishes

127 comprise the non-teleost lineages of polypteriformes (e.g. bichir), acipenseriformes (e.g.

128 sterlet sturgeon), holosteans (lepisosteiformes, e.g. spotted gar, and amiiformes, e.g.

129 bowfin), and the teleosts, subdivided into three major living lineages: osteoglossomorphs

130 (e.g. arowana, mooneyes, and the freshwater elephantfish), elopomorphs (e.g. eels and

131 relatives) and clupeocephalans (e.g. zebrafish and medaka) (see [21] for a recent

132 phylogeny of ray-finned fishes).

133 Our phylogenetic analysis confirmed that Nos1 is present in all species of jawed

134 vertebrates examined (Fig. 1a, green shading). In contrast, most fish lineages retained

135 Nos2, including chondrichthyans (Callorhinchus milii, Rhincodon typus, Chiloscyllium

136 punctatum, and Scyliorhinus torazame), polypteriformes (Polypterus senegalus,

137 Erpetoichthys calabaricus), acipenseriformes (Acipenser ruthenus), holosteans (Amia

138 calva, Lepisosteus oculatus), elopomorphs (Megalops cyprinoides), osteoglossomorphs

139 (Paramormyrops kingsleyae, Scleropages formosus) and coelacanthiformes (Latimeria

140 chalumnae) (Fig. 1a, grey shading), although a nos2 gene loss event occurred at the stem

141 of Neoteleostei (Fig. 1b), since this gene has not been found in any available genome from

142 this clade. On the other hand, our phylogenetic analysis highlights the occurrence of extra

143 nos2 duplicates in several lineages, for which we adopted a specific nomenclature: in the

144 zebrafish Danio rerio there are two nos2 genes, nos2a and nos2b, while in the goldfish

145 Carassius auratus, the blind golden-line barbel Sinocyclocheilus anshuiensis and the

146 common carp Cyprinus carpio we found three: nos2a, nos2ba, and nos2bb; in salmonids

147 (Salmo salar and Oncorhynchus mykiss) there are two different copies of nos2, named

148 nos2α and nos2β; and last, we named nos2.1 and nos2.2 the two nos2 paralogs that we

149 found in a characid (the Mexican tetra Astyanax mexicanus), a gymnotid (the electric eel

150 Electrophorus electricus), an ictalurid (the channel catfish Ictalurus punctatus), an esocid

151 (the northern pike Esox lucius), and a clupeid (the Atlantic herring Clupea harengus) (Fig.

152 1a, grey shading). Our nomenclature is based both on the phylogenetic analysis and a

153 synteny conservation analysis (see below and in the Discussion section).

154 Nos3 deserves special attention since it was previously believed that a loss event

155 predated the lineage of actinopterygians or alternatively that it represents an innovation of

156 tetrapods [8]. Nevertheless, this latter hypothesis may have been overinterpreted since

157 few ray-finned genome sequences were originally available. The only actinopterygian nos3

158 gene reported thus far is in the spotted gar [20]. Here we report the identification of nos3

159 genes in genomes of the bichir P. senegalus, the sterlet sturgeon A. ruthenus [22], the

160 bowfin A. calva [23], and the freshwater elephantfish P. kingsleyae [24] (Fig. 1a, red

161 shading). The absence of nos3 in all available clupeocephalans indicates a gene loss

162 event in the stem of this group (Fig. 1c). Furthermore, we did not find nos3 in the tarpon M.

163 cyprinoides, the most complete genome available among Elopomorpha, nor in

164 transcriptomic data of the European eel Anguilla anguilla. On the other hand, we did

165 identify a nos3 ortholog in the cloudy catshark S. torazame, suggesting its presence in the

166 ancestor of gnathostomes. Previously, two nos genes had been found in the lamprey,

167 called nosA and nosB [8], with unresolved orthology to gnathostome nos1-nos2-nos3, and

168 derived from a lineage-specific tandem duplication in the lamprey lineage. Based on this

169 finding, we searched for the presence of nos genes in other cyclostomes. In the genome of

170 the arctic lamprey Lethenteron camtschaticum [25] we found orthologous genes to P.

171 marinus nosA and nosB paralogs. On the other hand, in the inshore hagfish Eptatretus

172 burgeri we identified a single nos gene. Our phylogenetic analysis shows that the hagfish

173 Nos remains outside lamprey NosA-NosB clade, therefore with no clear orthology

174 relationship to any specific gnathostome Nos1, Nos2, Nos3, and suggesting that the

175 duplication giving rise to the lamprey nosA-nosB occurred at least before the last common

176 ancestor of Petromyzontidae.

177

178 Figure 1. Evolution of the Nos gene family. a, Bayesian inference phylogenetic analysis 179 of Nos proteins in chordates. Nos1 clade is indicated by a green shading; light grey 180 shading for the Nos2 clade, and red shading for the Nos3 clade. Numbers at nodes 181 represent posterior probability values. Nos proteins from invertebrate chordates, the 182 lancelet Branchiostoma lanceolatum, and the tunicate Ciona robusta, were used as

183 outgroup sequences. b-c, Evolutionary scenarios indicating the loss of Nos2 event in 184 Neoteleostei (b) and Nos3 in Clupeocephala (c) as grey lines. Nos3 in Elopomorpha is 185 absent although parsimony suggests it was present in stem elopomorphs, and it is 186 indicated with a dashed line. The schematic representation of Nos1 was omitted because 187 it is present in single copy in all analysed gnathostome species. TGD stands for Teleost- 188 specific Genome Duplication. 189

190

191 In order to better understand the gene loss and expansion events depicted by our

192 phylogenetic analysis, we next analysed the microsynteny (genes linked in proximity) of

193 nos genes in different species. This revealed a complex evolutionary scenario for nos2

194 compared to nos1 and nos3. Specific nos2 duplications in different lineages are explained

195 by distinct evolutionary events in teleosts (Fig. 2a and Supplementary Figure 1). First, the

196 lack of synteny conservation between nos2a and nos2b in cyprinids, and the lack of nos2a

197 in the expected location in non-cyprinid fishes (Supplementary Figure 1) indicates that

198 these paralogs originated in a specific gene duplication event in a common ancestor of the

199 lineage, independently from the TGD (the alternative explanation would require numerous

200 nos2a losses in several fish lineages), in which while nos2b has remained the ancestral

201 genomic location, nos2a has been translocated to a different position in the genome (Fig.

202 2a and Supplementary Fig. 1). Second, an additional genome duplication event after the

203 TGD specifically occurred independently in several teleost lineages, causing the presence

204 of extra nos2 paralogs. These include some cyprinids, in which a carp-specific genome

205 duplication event (Cs4R) likely occurred before the divergence of C. auratus, S.

206 anshuiensis and C. carpio [26], and salmonids (salmonid-specific genome duplication or

207 Ss4R) [27,28], with S. salar and O. mykiss in this study. These additional tetraploidization

208 events can explain the origin of the two independent sets of nos2 genes in cyprinid and

209 salmonid species. In the case of cyprinids, both our phylogenetic and synteny analyses

210 clearly show their nos2b orthology, and we denote them as nos2ba and nos2bb (Fig. 1a

211 and Fig. 2a). In the case of salmonids, we name them nos2α and nos2β to distinguish

212 them from the cyprinid nos2a and nos2b paralogs, which have a separate origin (see

213 above; Fig. 2a). Third, independent tandem gene duplications explain the presence of two

214 nos2 copies, that we named nos2.1 and nos2.2, located next to each other in the same

215 chromosomal fragment in the genomes of the Atlantic herring (C. harengus), the Mexican

216 tetra (cavefish, A. mexicanus), the electric eel (E. electricus), the channel catfish (I.

217 punctatus), and the northern pike (E. lucius) (Fig. 2a).

218 Bichir, reedfish, sterlet, spotted gar, bowfin and freshwater elephantfish are the only ray-

219 finned fishes that retained a nos3 ortholog. Therefore, we investigated the absence of

220 nos3 in clupeocephalans. First, we looked for the genomic region containing nos3 in fishes

221 that represent outgroups to the clupeocephalans. We found one long scaffold of the P.

222 kingsleyae genome (scaffold 217) [24] showing extensive conserved synteny with the

223 nos3-containing segment of the linkage group 11 (LG) in the spotted gar genome (Fig. 2b).

224 While these appear to correspond to one of the TGD ohnologons (Fig. 2b), there are other

225 two P. kingsleyae scaffold segments (from scaffolds 72 and 104) that together seem to

226 represent the second TGD ohnologon, but lacking the expected nos3 TGD ohnolog (Fig.

227 2b). Zebrafish chromosomes 16 and 19 and medaka chromosomes 11 and 16 contain

228 orthologous regions to the two P. kingsleyae and L. oculatus TGD ohnologons, but lack a

229 nos3 gene at the expected locations. One-to-one relationship between these P. kingsleyae

230 scaffolds and zebrafish and medaka chromosomes is challenging to determine (Fig. 2b).

231 Regardless, the most parsimonious explanation for the nos3 repertoire in ray-finned fishes

232 is that, first, one of the two nos3 TGD ohnologs was lost in the teleost common ancestor,

233 while the other was retained and later lost in secondary, independent events in the

234 common ancestor of Clupeocephala and, probably, that of Elopomorpha (Fig. 1c and Fig.

235 2b).

236

237 Figure 2. Conserved microsynteny of nos2 and nos3. a, The nos2 paralogs derived 238 from different duplication modalities: carp-specific genome duplication (Cs4R) (nos2ba and 239 nos2bb in the goldfish and blind golden-line barbel); salmonid-specific genome duplication 240 (Ss4R) (nos2α and nos2β in the Atlantic salmon and rainbow trout); tandem gene 241 duplication occurred independently in five lineages (nos2.1 and nos2.2 in the northern 242 pike, Atlantic herring, electric eel, Mexican tetra and channel catfish). An additional nos2 243 duplicate (nos2a) is present in cyprinids (zebrafish, goldfish, and blind barbel). b, A 244 conserved synteny map of genomic regions around the nos3 gene locus highlights the loss 245 in Clupeocephala (including zebrafish and medaka), and in Osteoglossomorpha

246 (arowana). Genes are represented as arrows and are coulor coded according to their 247 orthology and ohnology. The direction of arrows indicates transcription orientation. The 248 symbol // indicates a long-distance on a chromosome. The asterisk indicates scaffold 72 of 249 the freshwater elephantfish genome [24]. 250

251

252 Expression of nos in vertebrate developing gills

253 Spotted gar is an important emerging model organism because it represents an

254 evolutionary bridge between teleosts and tetrapods that facilitates cross-species

255 comparisons. The gar genome is slowly evolving compared to that of teleosts and has

256 preserved a more ancient structural organization [29]. Therefore, we examined the

257 expression patterns of nos genes during gar development. As expected, based from the

258 literature, nos1 was expressed in several regions of the developing nervous system

259 (Supplementary Fig. 2). In contrast, nos2 expression was not detected during the

260 developmental stages covered in the present study, i.e., from 4 to 14 days post fertilization

261 (dpf). Unexpectedly, the expression of nos3 was first detected in gar embryos in the

262 pharyngeal area at 4 dpf (Fig. 3a-b) and increased at 6 dpf (Fig. 3c-d). At 7 dpf, embryos

263 showed clear nos3 expression in developing arches III, IV, and V (Fig. 3e-g). Later, at 11

264 dpf, the positive signal is localized in gill filaments (Fig. 3i-k). Histological sections

265 highlighted the presence of nos3 in the epithelium of branchial lamellae (Fig. 3l), also

266 confirmed by the signal in gill structures in an advanced stage of maturation in 14 dpf

267 juveniles (Fig. 3m-p).

268

269

270 Figure 3. Spotted gar nos3 localization during development. Expression of nos3 is 271 localized in the pharyngeal area in 4 dpf (a-b) and 6 dpf (c-d) embryos, in pharyngeal 272 arches in 7 dpf larvae (e-g) schematized in (h), in developing gills in 11 dpf late larvae (i-l), 273 and in gill lamellae in 14 dpf juveniles (m-p). Coronal (n) and transversal section (o) 274 planes are indicated with a red dashed line in (m). Abbreviations: ey, eye; gi, gill; dv, 275 dorsal view; lv, lateral view. Scale bar is 1 mm in a, c, e, i, m; 100 µm in b, d, l, n, o, p. 276

277

278 The detection of nos3 transcripts in gills of spotted gar and the established involvement of

279 NO gas in osmoregulatory control and vascular motility in gills of numerous teleosts [30–

280 35] prompted us to investigate whether a similar nos expression patterns occurred in

281 developing gills of other fish species. We investigated nos expression in the sterlet

282 sturgeon A. ruthenus and the bichir P. senegalus, members of early-branching groups of

283 ray-finned fishes [21]. Moreover, we similarly investigated nos expression in the

284 chondrichthyan cloudy catshark S. torazame to infer the ancestral expression condition

285 among gnathostomes. Unlike gar, we discovered that nos3 was not expressed in gills of

286 other species analysed in this work (Supplementary Fig. 2), thus raising questions about

287 whether nos3 expression in gills represents an oddity of holosteans or gars. Surprisingly,

288 nos1 and nos2 were expressed in gills of sturgeon, bichir, and shark. In particular, nos2

289 was expressed in the branchial area of the sterlet sturgeon (Fig. 4a-c) and bichir embryos

290 (Fig. 4d-f), while nos1 is expressed in gills of catshark embryos (Fig. 4g-i).

291

292

293 Figure 4. Expression of nos genes in developing gills of sterlet sturgeon, bichir, and 294 shark embryos. The expression of nos2 in the gills of sterlet sturgeon Acipenser ruthenus 295 (14 mm stage, a-c) and bichir Polypterus senegalus (stage 31, d-e); nos1 in the shark 296 Scyliorhinus torazame (stage 27, g-i). Higher magnification views of the gill structure of a, 297 d, g are shown in b, e, h, respectively. The arrowheads indicate sectioning plane (a, d, g):

298 transversal sections (c, f, 50 µm) and frontal section (I, 10 µm). Abbreviations: gi, gill; yo, 299 yolk; pf, pectoral fin. Scale bar in a, d, g is 0.5 mm. 300

301

302 Our results show that nos paralogs are expressed in pharyngeal arches and gills in both

303 actinopterygians and chondrichthyans. These findings lead us to question whether nos

304 expression in gills could be a conserved feature also in sarcopterygians, and in particular

305 in amphibians that use gills for gas exchange. Therefore, to investigate the presence of

306 nos transcripts in amphibia, we chose the neotenic axolotl Ambystoma mexicanum

307 because it retains functional external gills throughout life. Gene expression analysis by

308 qPCR revealed that nos1 and nos2 are almost not detectable in adult axolotl gills, while

309 nos3 turned out to be highly expressed in gill structures (Supplementary Fig. 3). Therefore,

310 we conclude that nos expression in gills is a conserved feature in neotenic amphibian

311 assayed, previously observed exclusively in fishes.

312

313 Expression of nos genes in the lamprey

314 In cyclostomes (jawless vertebrates, including lampreys and hagfish), cartilaginous and

315 bony gnathostomes (jawed vertebrates), gills are endoderm-derived structures, pointing to

316 a single origin of pharyngeal gills before the divergence of these vertebrate lineages

317 [36,37]. The two lamprey nos paralogs, nosA, and nosB, display an unresolved orthology

318 relationship with their gnathostomes nos1, nos2, and nos3 (Fig. 1). To assess whether

319 nosA and nosB are expressed in gills during embryogenesis, we performed whole-mount

320 in situ hybridization experiments at different embryonic stages. We found that lamprey

321 nosA was expressed in several tissues, including the brain, dorsal midline epidermis,

322 tailbud, mouth, and cloaca, but not in gills (Fig. 5a-b). Conversely, the lamprey nosB

323 paralog showed restricted expression in the developing mouth, specifically in the cheek

324 process, including upper and lower lip regions (Fig. 5c-d). These results show that in the

325 arctic lamprey, neither of the two nos paralogs is expressed in immature or mature gills,

326 suggesting a fundamental difference in the role of nos genes in jawless and jawed

327 vertebrates.

328

329

330 Figure 5. Expression patterns of nosA and nosB in larvae of the arctic lamprey. At 331 stage 24-25 the nosA is expressed in the brain, mouth, upper and lower lip, dorsal midline 332 epidermis, and cloaca (a). At stage 28, nosA expression is restricted to the mouth (b). The 333 nosB is exclusively expressed in the cheek process, consisting of upper and lower lips (c- 334 d), and faint expression in the dorsal midline epidermis (c). Abbreviations: br, brain; cl, 335 cloaca; dme, dorsal midline epidermis; gp, gill pouches; mo, mouth; ll, lower lip; ul, upper 336 lip; lv, lateral view; vv, ventral view. Scale bar in a is 0.5 mm. 337

338

339 Discussion

340 Actinopterygian fishes experienced one of the largest radiations in the animal kingdom and

341 their history represents a valuable resource for the formulation of hypotheses regarding

342 the evolution of vertebrate gene families. In this work, we employed data from recent

343 genome projects to clarify and update the evolution of Nos family across vertebrates. We

344 performed a phylogenetic reconstruction using Nos protein sequences from key vertebrate

345 groups, including cyclostomes for which little information has previously been available.

346 Our phylogenetic analysis confirmed that Nos1 is ubiquitously present as single copy gene

347 across the gnathostome lineage, at least in the covered osteichthyan and chondrichthyan

348 species. Branch lengths of the Nos1 clade suggest a slow evolutionary rate throughout

349 vertebrate evolution in respect to the other two nos genes. Furthermore, our phylogenetic

350 data, complemented with syntenic analyses, highlighted for the first time a highly complex

351 scenario of Nos2 evolution, for which we suggest a nomenclature that attempts to

352 incorporate evolutionary origins into gene names. Previous analyses showed the presence

353 of two nos2 genes (nos2a and nos2b) in zebrafish and goldfish [38,39]. Here we show the

354 presence of a nos2a paralog also in other two cyprinids, C. carpio and S. anshuiensis (Fig.

355 1a and Fig. 2a). nos2a and nos2b likely derive from an event of gene duplication that

356 occurred specifically at the stem of the group, and not related to the classic TGD. This

357 result is supported by synteny analysis since the chromosomal position of nos2a and

358 nos2b genes is not conserved (Fig. 2a and Supplementary Fig. 1), as it would be expected

359 if they were retained after a whole-genome duplication. On the other hand, the cyprinid

360 nos2b paralog independently duplicated in carps after the Cs4R [26], as the conserved

361 synteny suggests (Fig. 2a). In salmonids, synteny analysis also implies that the two Nos2

362 paralogs originated secondarily after the Ss4R (Fig. 2a) [27,28]. Here, we call these genes

363 nos2ba and nos2bb in carps to emphasize and clarify their relationships to zebrafish

364 genes, and nos2α and nos2β in salmonids to indicate their distinct evolutionary origin.

365 Additionally, the present work shows that nos2 has undergone several independent

366 lineage-specific tandem gene duplication events (nos2.1 and nos2.2) (Fig. 2a). The search

367 of nos2 in available fish genomes, covering all main groups, failed to find it in any

368 Neoteleostei, and for this reason, we hypothesized a nos2 gene loss event occurred in

369 stem Neoteleostei (Fig. 1 and Fig. 6). It is worth mentioning that NO produced upon

370 stimulation of the inducible nos (nos2) is considered one of the most versatile players of

371 the immune system against infectious diseases, autoimmune processes and chronic

372 degenerative diseases [4,40]. For this reason, it would be important in the future to

373 investigate the impact of Nos2 loss on the immune response in Neoteleostei and if any

374 compensatory mechanisms occurred through the activation of other nos paralogs. In

375 addition, Nos2 is the only nos gene with retained duplicates in vertebrates, therefore, it

376 would also be important to understand if nos2 duplicates underwent neofunctionalization

377 or subfunctionalization, thus providing new functional features to the organism.

378 Concerning nos3, our understanding of its evolutionary history had a twist with the finding

379 of a nos3 ortholog in the spotted gar genome [20], proving that the previously postulated

380 actinopterygian-specific loss of nos3 was an incorrect inference. Fostered by this

381 discovery, we specifically searched for the presence of nos3 orthologs in a wide range of

382 fish species to infer the ancestral condition. We identified a nos3 gene in bowfin, thus

383 confirming the presence of nos3 in the other reference genus of the holostean clade, in

384 addition to gar (Fig. 1a and Fig. 6). Furthermore, the presence of nos3 in genomes of

385 bichir and sterlet sturgeon, which diverged prior to the teleostean and holostean split,

386 confirmed the hypothesis that nos3 was already present in the common ancestor of extant

387 osteichthyans, rather than an innovation of tetrapods [8] or neopterygians (holosteans plus

388 teleosts) [20] (Fig. 6). We did not find nos3 gene in the tarpon M. cyprinoides genome (Fig.

389 2b), and to date, the limited genomic and transcriptomic data of eels, congers, and morays

390 cannot endorse the presence of a nos3 in Elopomorpha. Therefore, more genome

391 sequences are necessary to confirm its absence in this key group. We also did not find

392 nos3 in any fish from Clupeocepahala (non-elopomorph and non-osteoglossomorph

393 teleosts; Fig. 1a and Fig. 2b) suggesting that a loss event took place in the common

394 ancestor of clupeocephalans (Fig. 1c and Fig. 2b). Notably, we found a nos3 gene in the

395 osteoglossomorph elephantfish P. kingsleyae (Fig. 1a and Fig. 2b), and it allowed us to

396 confirm that the loss of nos3 did not occur in the last common teleost ancestor, as

397 previously thought [20]. These findings suggest instead the following evolutionary scenario

398 for the nos3 gene: first, since we only find a maximum of one nos3 gene in those cases

399 where it is present, we assume that one of the two TGD ohnologs was immediately lost

400 after the TGD, and the other one was retained. This nos3 gene was then lost in the

401 ancestors of elopomorphs –although further research is needed to confirm this– and

402 clupeocephalans independently in separate events (Fig. 6).

403 The discovery of nos3 in sharks (S. torazame in this study) suggests that the origin of nos3

404 predates the divergence of gnathostomes and that three distinct nos paralogs were

405 already present in the last common ancestor of gnathostomes (Fig. 6), likely originating

406 after the two rounds of whole-genome duplication that took place during early vertebrate

407 evolution (VGD1 and VGD2, 2R hypothesis) [41]. The origin of nos genes is, in fact,

408 supported by the linkage to the evolutionarily conserved Hox gene clusters and several

409 other syntenic genes (Fig. 6b and Supplementary Fig. 4). Under this scenario, then a

410 fourth nos gene (putative nos4) should have existed but was apparently lost early in the

411 gnathostome evolution (Fig. 6a).

412 The apparent lack of nos genes in some vertebrate lineages remains to be clarified, such

413 as the absence of nos3 in coelacanth L. chalumnae (an extant basally diverging

414 sarcopterygian), in arowana S. formosus (an osteoglossomorph), and in elopomorph

415 fishes. In the future, further genomic projects will surely fill these gaps in our

416 understanding of this fascinating gene family.

417

418

419 Figure 6. Nos evolution in light of recent gene findings in vertebrates. The proposed 420 evolution of nos genes in gnathostomes (a) supposes an ancestral loss of a predicted 421 fourth nos gene (grey box), based on the linkage of human nos and Hox clusters (b). Loss 422 of nos3 occurred in stem Clupeocephala and loss of nos2 in stem Neoteleostei (a). 423 Species-specific nos2 duplications occurred in some Otomorpha, including Cyprinidae and 424 Characidae families. 425

426

427 The importance of NO in the ontogeny and function of vertebrate gills has already been

428 documented in the context of physio-pharmacological studies, primarily using inhibitors of

429 Nos activity. In gills, NO acts as a paracrine and endocrine vasoactive modulator and,

430 therefore, plays a crucial role in the distribution of oxygenated blood [42]. Moreover, NO

431 has an osmoregulatory function controlling the movement of ions across the gill epithelium

432 [33,43–45], and represents an important molecular component of the immune system

433 employed by macrophages to attack and destroy pathogens [46]. Nevertheless,

434 documentation of Nos enzymatic activity in fish gills has relied exclusively upon techniques

435 unable to discriminate among individual Nos proteins, such as NADPH-diaphorase activity

436 and immunolocalization with heterologous mammalian antibodies [42,44,45,47]. Therefore,

437 the detected enzymatic activity has for a long time been indicated generically as ‘Nos-like’.

438 Here we used a different approach based on mRNA transcript detection methodology,

439 which unequivocally distinguishes different genes, and showed, for the first time, that

440 indeed nos genes are expressed in gills during development in various vertebrates.

441 However, surprisingly different Nos paralogs are expressed in gills in different animals

442 tested: nos1 in shark, nos2 in bichir and sterlet sturgeon, and nos3 in spotted gar. The

443 most parsimonious hypothesis to explain this result is that the ancestral nos gene had a

444 number of roles in gills, immune system, brain, and other organs that was controlled by

445 separate regulatory elements and, due to subfunctionalization after the vertebrate 2R

446 (according to the Duplication-Degeneration-Complementation (DDC) model) [48], these

447 physiological roles partitioned to different nos ohnologs as lineages diverged and

448 reciprocal loss of the gill expression function occurred in a lineage-specific way. Further

449 support to this hypothesis comes from the identification of nos1-positive cells in gill of

450 zebrafish at 5 dpf, in addition to brain, eye, periderm and NaK ionocytes, according to the

451 recently released developmental single-cell transcriptome atlas [49] (Supplementary Fig.

452 5).

453 Additionally, to corroborate the involvement of NO in normal gill physiology, we searched

454 for nos expression in gills of a paedomorphic amphibian, the Mexican axolotl, which

455 maintains gill structures in adulthood. Taking into account the different evolutionary and

456 developmental origin of internal and external gills [50], the conservation of nos3

457 expression in gills indicated that the NO signaling system could be indeed fundamental for

458 the physiology and development of this structure in the axolotl, and perhaps generally in

459 pre-metamorphic amphibians. Therefore, our data obtained from established and

460 emerging model species highlighted that the expression of at least one nos gene has a

461 functional role in gnathostome gills.

462 Recently, a single origin of pharyngeal gills predating the divergence of cyclostomes and

463 gnathostomes was suggested [36]. Therefore, we investigated whether either of the two

464 arctic lamprey nos paralogs is expressed in developing gills, but found them expressed

465 mainly in the nervous system, mouth and pharynx, similarly to the expression pattern

466 previously reported in the cephalochordate amphioxus [51,52]. Nevertheless, we found

467 neither of the two genes to be expressed in gills during lamprey development, leading us

468 to speculate that either the expression of nos genes in gills was acquired in gnathostomes

469 after the divergence from cyclostomes, or alternatively, gill expression was a feature of

470 their last common ancestor but lost in the lamprey lineage. Future work on hagfish

471 embryology would be necessary to help solve this issue.

472 In conclusion, our findings pave the way for future studies that aim to investigate the

473 ontogenetic role of nitric oxide in gill development of aquatic vertebrates, possibly by loss-

474 of-function approaches using either Nos protein-specific chemical inhibitors or CRISPR-

475 Cas9 gene editing. From the perspective of the evolution of developmental mechanisms, it

476 would be interesting to understand more about the species-specific regulatory

477 mechanisms that drive different nos genes expression patterns in gills in different species

478

479

480 Materials and Methods

481 Sequence mining and phylogenetic analysis

482 Nos sequences used for evolutionary analyses were retrieved from NCBI

483 (https://www.ncbi.nlm.nih.gov/), Ensembl (www.ensembl.org/index.html), Skatebase

484 (http://skatebase.org/) and DDBJ (https://www.ddbj.nig.ac.jp/index-e.html) databases,

485 using direct browser webpages or by downloading fully assembled genomes and

486 transcriptomes (see Supplementary Table 1 for accession numbers). The quality of protein

487 sequences was checked and, where needed, manually curated excluding from the dataset

488 partial or low blast score sequences. We used proteins from Homo sapiens, Anolis

489 carolinensis and Xenopus tropicalis as internal references, and two non-vertebrate

490 chordates as outgroups: the cephalochordate Branchiostoma lanceolatum NosA, NosB

491 and NosC, and the tunicate Ciona robusta Nos.

492 Nos sequences from bowfin (A. calva) were obtained from a draft genome assembly [23].

493 Lamprey nosA and nosB genes were obtained by TBLASTN v2.2.31+ searches [53] from

494 the v1.0 draft genome of arctic lamprey L. camtschaticum [25] and the germ line draft

495 genome of sea lamprey P. marinus [54]. Initial predictions were extended, corrected, and

496 confirmed by RACE PCRs in the case of L. camtschaticum. Both P. marinus nosA and

497 nosB were manually curated using Wise2 [55]. The single nos gene sequence from the

498 inshore hagfish E. burgeri was obtained from a de novo transcriptome assembly [56].

499 Sequences of nos1, nos2, and nos3 genes from the cloudy catshark S. torazame were

500 obtained from a de novo transcriptome assembly [56] employing TBLASTN v2.2.31+. A

501 partial nos1 sequence (g15096.t1) was found in the European eel A. anguilla

502 transcriptome database (EeelBase 2.0, http://compgen.bio.unipd.it/eeelbase/), but it was

503 deliberately excluded from the phylogenetic analyses because of alignment ambiguities,

504 probably due to gene assembly errors.

505 For phylogenetic analysis, Nos amino acid sequences were aligned using the MUSCLE

506 algorithm [57] as implemented in MEGAX (version 10.2.4) [58], with default parameters,

507 run in a MacOS 11.2.1 operating system and saved in FASTA format. The alignment was

508 trimmed by trimAl v1.2rev59 [59], using the ‘-automated1’ parameter. The trimmed

509 alignment was then formatted into a nexus file using readAl (bundled with the trimAl

510 package) (supplementary file S1). A Bayesian inference tree was constructed using

511 MrBayes v3.2.6 [60], under the assumption of an LG+I+G evolutionary model. Two

512 independent MrBayes runs of 2,000,000 generations were performed, with four chains

513 each and a temperature parameter value of 0.05. The tree was considered to have

514 reached convergence when the standard deviation stabilized under a value of <0.01. A

515 burn-in of 25% of the trees was performed to generate the consensus tree (1,500,000

516 post-burnt-in trees).

517

518 Synteny

519 Conserved synteny analyses were manually performed on fish chromosomes or scaffolds.

520 With the aim of finding synteny blocks flanking the nos2 and nos3 orthologs, we employed

521 the Synteny Database (http://syntenydb.uoregon.edu/synteny_db/) [61,62]. Additional

522 information was retrieved in NCBI (https://www.ncbi.nlm.nih.gov/), Ensemble v.102

523 (http://www.ensembl.org/index.html) and Genomicus v100.01

524 (https://www.genomicus.biologie.ens.fr/genomicus-100.01/cgi-bin/search.pl) [61].

525

526 Collection of embryos and tissues

527 Spotted gar L. oculatus adult specimens were collected from the Atchafalaya River basin,

528 Louisiana (USA) and cultured in a 2 m diameter tank containing artificial spawning

529 substrate. Spawning was induced by injection of Ovaprim© (0.5 ml/kg) and embryos were

530 raised in fish water (salinity 1 ppt) at 24°C in a 14/10 h light/dark cycle [63]. The

531 developmental staging was determined following hours or days post fertilization in addition

532 to morphological criteria [64].

533 Embryos of L. camtschaticum were obtained by artificial fertilization, cultured at a

534 temperature ranging between 9 and 12°C, and staged as previously described [65,66].

535 Embryos of S. torazame were obtained, cultured, and staged as previously described

536 [67,68].

537 Bichir P. senegalus embryos were obtained from the breeding colony at the Department of

538 Zoology, Charles University, Prague (Czech Republic) by natural breeding. Embryos were

539 kept at 28°C and staged using Diedhiou and Bartsch (2009) guidelines [69]. Sterlet

540 sturgeon A. ruthenus embryos were obtained from the hatcheries of the Research Institute

541 of Fish Culture and Hydrobiology in Vodnany, University of South Bohemia (Czech

542 Republic). Embryos were raised in tanks containing E2 Pen/Strep zebrafish medium and

543 incubated at 17°C until the desired stages, according to Dettlaff and collaborators (1993)

544 [70]. Gill tissues from two adult axolotls (RRID:AGSC_110A) were collected under

545 benzocaine anesthesia (University of Kentucky, USA, IACUC protocol 2017-2580).

546

547 Gene expression analysis by in situ hybridization

548 For all species used in the present study, total RNA was isolated from a mix of embryo

549 stages using the phenol-chloroform method with TRIzol (Thermo-Fisher Scientific). cDNA

550 was synthesized from 1 µg of total RNA using the SuperScript VILO cDNA Synthesis kit

551 (Thermo-Fisher Scientific). Primers for PCR amplification are listed in Supplementary

552 Table 2. Amplicons were cloned into the pGEM-T Easy Vector (Promega) and Sanger

553 sequenced. Antisense Digoxygenin-UTP riboprobes were synthesized using SP6 or T7

554 RNA polymerases and the DIG RNA Labeling kit (Roche).

555 Whole-mount in situ hybridization experiments were performed following protocols

556 previously described: spotted gar [71], bichir and sturgeon [72], lamprey [65], and shark

557 [73], with slight modifications. For spotted gar embryos at 7 dpf (Long & Ballard stage 24)

558 and 11 dpf (Long & Ballard stage 28), longer proteinase K (10 µg/mL) digestion times were

559 performed, respectively 25 and 35 minutes at 24°C. Moreover, endogenous melanin

560 pigment was removed using bleaching solution [(3% hydrogen peroxide (H2O2) and 1%

561 potassium hydroxide (KOH) in distillate water (ddH2O)] for a few minutes. For 14 dpf gar

562 embryos (Long & Ballard stage 31), we performed in situ hybridizations on cryosections,

563 as previously described [74], including modifications reported in [75].

564 Transversal vibratome sections of bichir and sturgeon embryos (thickness 50 µm) were

565 made on whole-mount hybridized embryos upon embedding in

566 gelatin/albumin/glutaraldehyde [50]. Shark embryos were embedded in paraffin after

567 whole-mount in situ hybridization assays, and frontal sections (10 µm) were obtained with

568 a microtome.

569 Whole-mount and sectioned preparations mounted on slides were imaged on Axio Imager

570 Z2 with Apotome 2 (Carl Zeiss), equipped with Axiocam 503 coulor digital camera and

571 Axio Vision software for analysis. Whole-mount bichir and sturgeon embryos were

572 photographed as Z-stacks using a motorized dissection microscope (Olympus SZX12) and

573 deep-focus images were generated by merging Z-stacks in QuickPhoto Micro.

574

575 Real-time PCR

576 Expression levels of nos genes in axolotl A. mexicanum gills were analysed by qPCR

577 using specific primers reported in Supplementary Table 2. The atpf51 gene was used as a

578 reference. RNA was isolated by performing a chloroform extraction and isopropanol

579 precipitation. RNA was quantified using a Nanodrop and 1 µg of total RNA was used to

580 generate cDNA using an Invitrogen Super-Script IV cDNA synthesis kit with oligo-dT

581 tailing. RT-qPCR was performed in triplicate with SYBER Master Mix on a LightCycler 96

582 (Bio-Rad), using a 2-step amputation protocol (95°C for 10 sec, 60°C for 30 sec) and 40

583 cycles. Data were analysed using the ΔΔCT method.

584

585 Data availability

586 Accession numbers of protein sequences used in the phylogenetic analysis are available

587 in Supplementary Table 1. Primer sequences used for the synthesis of in situ hybridization

588 riboprobes and in quantitative real-time PCR experiments are given in Supplementary

589 Table 2.

590

591 Acknowledgments

592 The authors thank Allyse Ferrara and Quenton Fontenot, Louisiana State University

593 (USA), for their help in the generation of spotted gar embryos. The authors thank Fumiaki

594 Sugahara for his help in the interpretation of results in the arctic lamprey, Anna Pospisilova

595 for technical assistance with bichir and sturgeon in situ hybridizations, and Martin

596 Psenicka, Roman Franek, Michaela Fucikova, Marek Rodina, David Gela, and Martin

597 Kahanec for sterlet sturgeon spawns. A special thanks to Robert Cerny for the

598 establishment of the African bichirs colony at the Charles University in Prague.

599 Giovanni Annona was supported by the Research grant POR Campania FSE 2014/2020

600 (IT) and by the EMBO Short Term Fellowship (# 6936) to visit the Postlethwait laboratory

601 in Oregon (USA) and for the field trip in Louisiana (USA). Jan Stundl is supported by the

602 European Union’s Horizon 2020 research and innovation program under the Marie

603 Skłodowska-Curie grant agreement No. 897949. Vladimir Soukup is supported by the

604 Charles University Research Centre program No. 204069 and grant SVV260571/2020.

605 Randal Voss and the Ambystoma Genetic Stock Center are supported by the National

606 Institutes of Health, USA (P40OD019794). John H. Postlethwait is supported by the R01

607 OD011116 grant from the US National Institutes of Health. Salvatore D’Aniello is

608 supported by the NOEVO grant from the Stazione Zoologica Anton Dohrn Napoli.

609

610 Competing interests

611 The authors declare no competing interests.

612

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